Field of the Invention
The present invention in the fields of molecular biology, immunology and medicine relates to chimeric nucleic acid molecules that encode an antigen, a signal peptide, and an immunogenicity-potentiating polypeptide (“IPP”) such as the heat shock protein HSP70, and their uses a immunogenic compositions to induce and enhance immune responses, primarily cytotoxic T lymphocyte responses to specific antigens such as tumor or viral antigens.
Description of the Background Art
Cytotoxic T lymphocytes (CTL) are critical effectors of anti-viral and antitumor responses (reviewed in Chen, C H et al., J Biomed Sci. 5: 231-252, 1998; Pardoll, D M. Nat Med. 4: 525-531, 1998; Wang, R F et al., Immunol Rev. 170: 85-100, 1999). Activated CTL are effector cells that mediate antitumor immunity by direct lysis of their target tumor cells or virus-infected cells and by releasing of cytokines that orchestrate immune and inflammatory responses that interfere with tumor growth or metastasis, or viral spread. Depletion of CD8+ CTL leads to the loss of antitumor effects of several cancer vaccines (Lin, K-Y et al., Canc Res. 56: 21-26, 1996; Chen, C-H et al., Canc Res. 60: 1035-42, 2000). Therefore, the enhancement of antigen presentation through the MHC class I pathway to CD8+ T cells has been a primary focus of cancer immunotherapy.
DNA vaccines have emerged as an attractive approach for antigen-specific cancer immunotherapy. DNA vaccines offer many advantages over more conventional vaccines, such as peptide or attenuated live pathogens. One advantage is that DNA vaccines are reasonably stable and can be easily prepared and harvested in large quantities. Additionally, naked plasmid DNA is relatively safe and can be repeatedly administered without adverse effects. Furthermore, because DNA is able to be maintained in cells for long-term expression of the encoded antigen, maintenance of immunologic memory is possible (for reviews, see Donnelly, J J et al., Annu Rev Immunol 1997, 15:617-648; Pardoll, D. M., Nat Med 1998, 4(5 Suppl):525-531; Robinson, H L, Vaccine 1997, 15:785-787; Gurunathan, S et al., Annu Rev Immunol 2000, 18:927-974).
However, one limitation of these vaccines is their lack of potency, since the DNA vaccine vectors generally do not have the intrinsic ability to be amplified and to spread in vivo as do some replicating viral vaccine vectors. Furthermore, some tumor antigens such as the E7 protein of human papillomavirus-16 (“HPV-16”) are weak immunogens (Chen et al., 2000, supra). Therefore, there is a need in the art for strategies to enhance DNA vaccine potency, particularly for more effective cancer and viral immunotherapy.
Heat Shock Proteins
Cells respond to stressors (typically heat shock) by increasing the expression of a group of genes commonly referred to as stress, or heat shock, genes. As used herein, a “heat shock protein” (abbreviated either HSP or Hsp) or “stress protein,” is encoded by a stress gene, and is therefore typically produced in significantly greater amounts upon the contact or exposure of the cell or organism to the stressor. A heat shock gene is a gene that is activated or otherwise detectably upregulated as a result of stressor exposure (which may include heat shock, hypoxia, glucose deprivation, a heavy metal salt, an inhibitor of energy metabolism and electron transport, and protein denaturant, or to certain benzoquinone ansamycins. See, for example, U.S. Pat. No. 6,524,825 and Nover, L., Heat Shock Response, CRC Press, Inc., Boca Raton, Fla. (1991), both of which are hereby incorporated by reference. Stress genes includes native homologues within known stress gene families, such as certain genes within the Hsp70 and Hsp90 stress gene families, even though not every homologue is induced by a stressor.
These proteins appear to participate in important cellular processes such as protein synthesis, intracellular trafficking, and assembly and disassembly of protein complexes. The increased amounts of stress proteins synthesized during stress serve primarily to minimize the consequences of induced protein unfolding. Indeed, the preexposure of cells to mildly stressful conditions that induce stress proteins protects the cells from the deleterious effects of more extreme forms of stress. The major stress proteins appear to be expressed in every organism and tissue type) examined so far. Moreover, they represent the most highly conserved group of proteins identified to date. For example, when stress proteins in widely diverse organisms are compared. Hsp90 and Hsp70 exhibit 50% or higher identity at the amino acid level and share many similarities at non-identical positions. Similar or greater homology exists between different members of a particular stress protein family within a species.
The predominant stress proteins in bacteria have molecular masses around 70 and 60 kDa, that are commonly referred to as Hsp70 and Hsp60, respectively. These represent about 1-3% of the total cell protein but accumulate to levels as high as 25% under stressful conditions.
Genes encoding stress proteins may be present in single or multiple, non-identical copies in a genome. For example, the human genome has at least one copy of an hsp100 gene, at least two different hsp90 genes, up to ten hsp70 genes of which at least several are non-identical copies, several T complex genes (Tcp genes) and at least one gene encoding the related mitochondrial protein Hsp60, as well as at least three copies of small hsp genes encoding Hsps of 20-30 kDa. Most families of stress genes include at least one member whose expression is relatively high and is either entirely constitutive or only mildly inducible. Furthermore, several families of stress genes include members that are not up-regulated by heat but are by other signals, e.g. increased calcium levels.
The stress proteins, particularly Hsp70, Hsp60, Hsp20-30 and Hsp10, are among the major determinants recognized by the immune system in response to infection by M. tuberculosis and M. leprae (Young, R A et al., Cell, 1989, 50:5-8. Even healthy individuals with no history of mycobacterial infection or autoimmune disease carry T cells that recognize both bacterial and human Hsp60 epitopes; a considerable fraction of T cells expressing the γδ T cell receptor recognize both self and foreign stress proteins (O'Brien, R et al. Cell, 1989 57:664-674 (1989). The “system” recognizing Hsp epitopes is considered to be an “early defense system” against invading microorganisms (Murray, P J et al., J. Bacteriol. 174:4193-6 (1992)) and may be maintained by frequent stimulation by bacteria and viruses. The safety of stress proteins is demonstrated by the success and relative safety of BCG (Bacille Calmette Guerin, a strain of M. bovis) vaccination, which induce an immune response against stress proteins that is cross-protective against M. tuberculosis. 
Immunogenic Constructs with HPV E7 as a Model Antigen
The present inventors and their colleagues previously developed several intracellular targeting and intercellular spreading strategies to enhance DNA vaccine potency using various IPP's (Hung, C F et al., J Virol 2002, 76:2676-2682; Cheng, W F et al., J Clin Invest 2001, 108:669-678; Hung, C F et al., J Immunol 2001, 166:5733-5740; Chen, C H et al., Gene Ther 1999, 6:1972-1981; Ji, H et al., Hum Gene Ther 1999, 10:2727-2740; Chen, C H et al., Cancer Res 2000, 60:1035-1042; U.S. Pat. No. 6,734,173, WO 01/29233; WO03/085085; WO 02/012281; WO 02/061113, etc.). Among these strategies, the linkage of Mycobacterium tuberculosis heat shock protein 70 (HSP70) to human papillomavirus type 16 (HPV-16) E7 has been demonstrated to dramatically increase E7-specific CD8+ T cell precursors and enhance anti-tumor effects against an E7-expressing tumor (TC-1) in vaccinated mice. These discoveries followed the earlier finding that immunization with HSP complexes isolated from tumor or virus-infected cells potentiated anti-tumor immunity (Janetzki, S et al., 1998. J Immunother 21:269-76) or antiviral immunity (Heikema, A E et al., Immunol Lett 57:69-74) Immunogenic HSP-peptide complexes could be reconstituted in vitro by mixing the peptides with HSPs (Ciupitu, A M et al., 1998. J Exp Med 187:685-91). Furthermore, HSP-based protein vaccines have been created by fusing antigens to HSPs (Suzue, K et al., 1996. J Immunol 156:873-879). The results of these investigations point to HSPs one attractive candidate for use in immunotherapy. However, prior to the present inventors' work, HSP vaccines were peptide/protein-based vaccines.
Moreover, the present inventors and their colleagues were the first to provide naked DNA and self-replicating RNA vaccines that incorporated HSP70 and other immunogenicity-potentiating polypeptides. The present inventors and their colleagues also demonstrated that linking antigen to intracellular targeting moieties calreticulin (CRT), domain II of Pseudomonas aeruginosa exotoxin A (ETA(dII)), or the sorting signal of the lysosome-associated membrane protein type 1 (Sig/LAMP-1) enhanced DNA vaccine potency compared to compositions comprising only DNA encoding the antigen of interest. To enhance MHC class II antigen processing, one of the present inventors and colleagues (Lin, K Y et al., 1996, Canc Res 56: 21-26) linked the sorting signals of the lysosome-associated membrane protein (LAMP-1) to the cytoplasmic/nuclear human papilloma virus (HPV-16) E7 antigen, creating a chimera (Sig/E7/LAMP-1). Expression of this chimera in vitro and in vivo with a recombinant vaccinia vector had targeted E7 to endosomal and lysosomal compartments and enhanced MHC class II presentation to CD4+ T cells. This vector was found to induce in vivo protection against an E7+ tumor, TC-1 so that 80% of mice vaccinated with the chimeric Sig/E7/LAMP1 vaccinia remained tumor free 3 months after tumor injection. Treatment with the Sig/E7/LAMP-1 vaccinia vaccine cured mice with small established TC-1 tumors, whereas the wild-type E7-vaccinia showed no effect on this established tumor burden. These findings point to the importance of adding an “element” to an antigenic composition to enhance in vivo potency of a recombinant vaccine: in this case, a polypeptide that rerouted a cytosolic tumor antigen to the endosomal/lysosomal compartment
Intradermal administration of DNA vaccines via gene gun in vivo have proven to be an effective means to deliver such vaccines into professional antigen-presenting cells (APCs), primarily dendritic cells (DCs), which function in the uptake, processing, and presentation of antigen to T cells. The interaction between APCs and T cells is crucial for developing a potent specific immune response.
However, the various DNA or RNA constructs described by the present inventors or others in the prior art, have resulted in certain combinations that induced a heightened immune response in experimental animals. However, none of these vaccines have been ideally designed use in humans where administration may be limited for practical or other reasons to intramuscular injection. Because direct transduction of professional APCs in muscle tissue is not likely to occur due to paucity of such cells in muscle. That leaves cross-priming as the most likely mechanism for the induction of heightened immunity in humans. Optimizing vaccine constructs for cross priming requires that an element be added that promotes the secretion of the expressed polypeptide antigenic moiety, preferably as a fusion polypeptide with a molecule that promotes antigen processing via the MHC class I pathway. Moreover, it best to used plasmid constructs that are know to be safe and effective in humans. Finally, in the case of HPV oncoprotein antigens, it is also important to “detoxify” the protein that is to be expressed so that it will not act as an oncogenic transforming agent. It is to such constructs with the aforementioned advantageous properties that the present invention is directed.